Journal of Cancer Therapy, 2011, 2, 394-400
doi:10.4236/jct.2011.23054 Published Online August 2011 (http://www.SciRP.org/journal/jct)
Selective Cytotoxicity and Apoptogenic Activity of
Hibiscus Sabdariffa Aqueous Extract against
MCF-7 Human Breast Cancer Cell Line
Shahnaz Khaghani1, Farideh Razi2, Mohsen Mohammadian Yajloo3, Malihe Paknejad1,4, Ahmad
Shariftabrizi1, Parvin Pasalar1,2*
1Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran; 2Endocrinology
and Metabolism Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran; 3Center for Research on Occupa-
tional Diseases (GRAD), Tehran University of Medical Sciences, Tehran, Iran; 4Students’ Scientific Research Center (SSRC), Te-
hran University of Medical Sciences, Tehran, Iran.
Received April 7th, 2011; revised June 3rd, 2011; accepted June 23rd, 2011.
ABSTRACT
Background: Herbal compounds are attractive anticancer candidates due to their low toxicity. Previous studies have
demonstrated that Hibiscus sabdariffa is promising as an anticancer agent against several cancer types; however, its
potential therapeutic role in breast cancer remains to be investigated. Materials and Methods: In the present study, the
cytotoxic effects of Hibiscus sabdariffa aqueous extract (HSE) on a human breast adenocarcinoma cell line (MCF-7)
and fetal foreskin fibroblast (HFFF) were investigated. Different concentrations of water extract of calyces were added
and the percentage of cell survival was determined after 24, 48, and 72 hours using an MTT assay. Apoptosis induction
was assessed by DNA fragmentation. Results: At the concentration of 0.5 mg/ml of the extract and following 72 hours
of incubation, the number of viable MCF-7 cells was less than 50%. The extract was not cytotoxic against normal
HFFF cells in all tested concentrations. Also, HSE induced apoptosis only in MCF-7 cells. Conclusions: These results
suggest that HSE inhibits the growth of MCF-7 cells selectively in a concentration- and time-dependent manner. As this
herbal substance has been shown to be nontoxic at very high doses in experimental animals, it might be a good anti-
cancer drug candidate for breast cancer treatment.
Keywords: Hibiscus Sabdariffa, Natural Anticancer Compound, MTT Assay, Apoptosis, Breast Cancer
1. Introduction
Cancer is a major public health problem in the United
States and many other parts of the world, accounting for
roughly 25% of total deaths annually. The three most
commonly diagnosed types of cancer among women in
2010 were cancers of the breast, lung, and colorectum,
accounting for 52% of cancer cases in this group. Breast
cancer alone accounted for 28% (207,090) of all new
cancer cases among women [1,2].
The toxicity associated with conventional cancer che-
motherapy arises primarily from the lack of specificity
for tumor cells. This leads to a low therapeutic index,
which results in unacceptable damage to normal organs
and consequently puts limitations on the dose of the drug
that can be administered. The majority of the currently
available anticancer drugs are designed to have selective
toxicity toward tumor cells [3,4]. Several approaches are
being considered to handle this problem and thus im-
prove the effectiveness and tumor-cell specificity of
anticancer drugs. Among these approaches, many studies
have focused on natural compounds that inhibit the
growth of cancer cells more selectively than normal cells.
Thus, phytochemicals have become an important cate-
gory of anticancer drugs. Over 75% of non-biological
anticancer drugs approved between 1981 and 2007 were
either natural products or were developed based on them
[5].
Herbal medicines are usually very easily accepted by
women suffering from breast cancer. As many as 80% of
women with breast cancer use some form of comple-
mentary or alternative medicine, the most common of
which is the use of herbs, in the hope that they might
lessen the side effects of treatment, improve quality of

Selective Cytotoxicity and Apoptogenic Activity of Hibiscus Sabdariffa Aqueous Extract Against MCF-7 Human 395
Breast Cancer Cell Line
life, provide a greater sense of control, and reduce stress
[6,7]. A beneficial effect of herbal substances in breast
cancer treatment is still in doubt mainly due to lack of
randomized clinical trials [8]. This is even true for Chi-
nese herbal rem e di es [9].
Hibiscus sabdariffa (also known as roselle, sour tea,
and karkade) from the Malvaceae family is cultivated
and naturalized in tropical and subtropical regions, in-
cluding south of Iran (Baluchestan). The plant has been
used in different countries as a culinary and medicinal
substance. The fleshy fruiting calyces of this plant, which
are sour in taste, have been utilized for preparing cand ies,
jellies, and hot and cold beverages. In folk medicine, the
calyx extract of this plant is used for treatment of several
illnesses, including high blood pressure, gastrointestinal
disorders, hypercholesterolemia, liver diseases, and fever
[10-13]. This plant has also been shown to have antican-
cer effects in vitro against gastric cancer [14,15], hepa-
tocellular cancer [16], and leukemia [17-19]. Hibiscus
protocatechuic acid has also been shown to inhibit the
carcinogenic effect of various chemicals in different tis-
sues of rats, including the liver, oral cavity, colon, glan-
dular stomach tissue, bladder, and skin [12]. The plant’s
extract is characterized by a very low degree of toxicity.
The LD50 of calyx extract in rats has been found to be
above 5000 mg/kg [12].
Constituents of Hibiscus sabdariffa have been exten-
sively studied since 1932. The chemicals present in the
flowers of Hibiscus sabdariffa include citric and malic
acids; tartaric acids; anthocyanins (delphinidin-
entoside-glucos i d e, d elphini din-3- glucoxyl oside, delp h i -
nidin-3-ambubioside, cyanidin-monoglucoside, cyanidin-
3-sambubioside, cyanidin-3,5-diglucoside, cyanidin-3-
glucosylrutinoside, cyanidin-3-glucoside); flavonol gly-
coside; gossypitrin; quercetin; myricetin; hibiscetrin;
sabdaretin; quercetin; luteolin, a luteolin glycoside and
chlorogenic acid; flavonoids (gossypetin, hibiscetin, and
their respective glycosides); protocatechuic acid; and
sterols (β-sitosterol and ergosterol). Different strains of
Hibiscus sabdariffa from different countries may differ in
one or several of these constituents [12].
The aim of the present study was to investigate the
cytotoxic effect of HSE on a human breast adenocarci-
noma cell line (MCF-7) and a human fetal foreskin fi-
broblast cell line (HFFF) as the benign counterpart.
2. Materials and Methods
2.1. Preparation of Aqueous Extract of Hibiscus
Sabdariffa
Hibiscus sabdariffa calyces were obtained in the tradi-
tional market in Tehran, Iran, and authenticated by the
Department of Botany, Tehran University of Medical
Sciences. The voucher specimen was deposited in the
herbarium of the Tehran University of Medical Sciences.
The samples were washed of any contaminants, then
dried and cr ushed. The powdered calyces were subj ected
to extraction by the maceration method with distilled
water at 25°C for 48 hours. The extract was filtered using
Whatman filter paper and then evaporated to dryness
using a rotary evaporator (40°C). The dried extract was
prepared as a stock solution, sterilized, and further di-
luted to final concentrations of 0.5, 0.4, 0.3, 0.2, 0.1, and
0.05 mg/ml.
2.2. Cell Culture
Human breast adenocarcinoma (MCF-7, NCBI C135)
and normal human fetal foreskin fibroblast (HFFF, NCBI
C170) cell lines, which were obtained from the National
Cell Bank of Iran (NCBI), were used in this study. These
cell lines were grown in a RPMI-1640 medium (Sigma,
USA), supplemented with 10% (V/V) fetal calf serum
(FCS) (Seromed, Germany), 1% of L-glutamine (2 mM)
(Sigma), 1% of sodium pyruvate (1 mM) (Sigma), and
1% of penicillin/streptomycin (50 IU/ml and 500 µg/ml,
respectively) (Sigma), according to the manufacturers’
instructions. The cell lines were maintained in a humidi-
fied atmosphere of 5% CO2 at 37°C. After the expansion
of cultures to approximately 75% - 80% confluency, cells
were trypsinized with 0.25% trypsin/0.04% EDTA solu-
tion in phosphate-buffered saline (PBS) (Sigma) for 1-2
minutes. For cytotoxicity assays, 2.5 × 104 cells/well
were cultured in 96-well microplates [9].
2.3. Cytotoxicity Assay
The cytotoxic effect of HSE against tumor and normal
cell lines was determined by a rapid colorimetric assay
using MTT (methyl-thiazolyl-tetrazolium bromide), and
results were compared with control [10]. In this assay,
mitochondrial dehydrogenase activity of viable cells re-
duces the soluble MTT into an insoluble colored forma-
zan product, which in turn can be dissolved in DMSO
(dimethyl sulfoxide) and measured spectrophotometri-
cally [11]. Briefly, 2.5 × 104 cells/well were seeded in 96
well microplates and incubated for 12 hours (37°C, hu-
midified air and 5% CO2). Then various concentrations
of the extract (0.5, 0.4, 0.3, 0.2, 0.1 and 0.05 mg/ml)
were added, and the microplates were further incubated
for 24, 48, and 72 hours in the same conditions. One
column of each microplate was used as negative control
(containing no extract). To evaluate the cell survival,
each well was incubated with 20 µl of MTT solution (5
mg/ml PBS) for 3 hours, and then 150 µl of the media of
each well was replaced with DMSO and mixed to dis-
solve insoluble formazan crystals. Then the absorbance

Selective Cytotoxicity and Apoptogenic Activity of Hibiscus Sabdariffa Aqueous Extract Against MCF-7 Human
Breast Cancer Cell Line
396
of each well was measured spectrophotometrically at 540
nm, and cell survival was calculated. Cytotoxicity was
considered significant whenever cell survival percentage
was below 50.
2.4. DNA Ladder Assay for Apoptosis
Cultured cells grown on a 100 mm Petri dish were
treated with various concentrations of HSE for 72 hours.
The cells were harvested by scraping and then centri-
fuged at 800× g for 10 minutes at 4°C. The pattern of
DNA cleavage was analyzed by agarose gel electropho-
resis using a DNA ladder kit (Roche, Germany) accord-
ing to the manufacturer’s instruction. Briefly, cells were
first lysed in a solution containing 6M guanidine-HCl,
10mM Urea, 10 mM Tris-HCL, 20% Triton X-100, and
pH = 4.4, followed by incubation for 10 minutes at 25°C.
Ispropropanol was added to the mixture, filtered, and
washed with a buffer containing 20 mM NaCl, 2 mM
Tris-HCl, and 80% ethanol. The DNA bound to the fil-
ters was finally eluted with a prewarmed elution buffer
(10 mM Tris, pH = 8.5). The sample DNA was sus-
pended in TAE buffer (10 mMTris–HCl and 0.5
mMEDTA, pH 8.0) and loaded onto a 1.2% agarose gel.
After electrophoresis, DNA was visualized by ethidium
bromide staining.
2.5. Statistical Analysis
Statistical analysis was performed using ANOVA and
postHOC tukey test using SPSS software. Significant
differences were established at p < 0.05.
3. Results
3.1. Selective Cytotoxic Activity of HSE against
MCF-7 and HFFF Cell Lines
Aqueous extract of Hibiscus sabdariffa appeared to be
toxic towards MCF-7 cells in a dose- and time-dependent
manner. Each concentration was assayed in eight wells
on each plate, and the average was considered as a single
experiment. Each experiment was repeated three times.
Cell survival was calculated by the following formula:
mean of test sample divided by mean of untreated sample
(negative control) in which cell survival percent was
taken as 100. Cytotoxicity was considered significant
whenever cell survival was less than 50%. The effect of
various concentrations and incubation times is depicted
in Figures 1(a)-(c). No significant cytotoxic effect was
observed in the MCF-7 cell line after 24 hours in all
concentrations. There was significant reduction of cell
numbers in the MCF-7 cell line following 48 and 72
hours in the doses of 0.4 mg/ml and above. At a concen-
tration of 0.5 mg/ml of the extract, following 72 hours of
incubation, cell survival was 45.51% (p < 0.05). To de-
termine whether this effect was selective, the extract was
also used against a human fetal foreskin fibroblast cell
line. The results indicated that unlike cancer cells, the
extract was not cytotoxic towards the HFFF cell line after
24 - 72 hours of incubation in all tested concentrations.
3.2. Apoptogenic Activity of HSE against MCF-7
Cell Line
Apoptogenic activity of HSE was assayed using a DNA
fragmentation method. Nuclear DNA fragmentation is a
biochemical hallmark of apoptosis, which shows DNA
fragments in multiples of 1 80 - 200 base pairs o n agaro se
gel, as opposed to the smear formation in necrotic cells.
The fragments correspon d in size to strands of DNA that
were cleaved at internucleosomal regions. These result
from activation of Caspase-3 and endonucleases. Thus,
gel electrophoresis was used to determine the mode of
death induction in MCF-7 and HFFF cell lines by HSE.
At the dose of 0.5 mg/ml following 72 hours of incuba-
tion, clear formation of a DNA ladder was observed
(Figure 2). No ladder formation was observed in HFFF
cells.
4. Discussion
The toxicity associated with cancer chemotherapy arises
primarily from the lack of specificity for tumor cells. To
handle this problem, the focus of many studies is on
natural compounds that inhibit cancer cells more selec-
tively than normal cells. This study was undertaken to
demonstrate the effects of HSE on MCF-7 and normal
HFFF cell lines. Here, we determined the cytotoxic con-
centration in which the plant extract significantly inhib-
ited growth of breast cancer cells. The same dose also
proved to be apoptogenic for the MCF-7 breast carci-
noma cell line.
Although the exact mechanisms of HSE aqueous ex-
tract on cancer cells are not known, mechanisms of
apoptosis induction in other models have been elucidated.
In gastric carcinoma, HSE induces apoptosis by p38
MAPK and JNK activation, and translocation of cyto-
chrome c from the mitochondria to the cytosol and cas-
pase cascade activation [14]. Polyphenol-rich HSE in-
duces apoptosis in gastric carcinoma cells by activation
of p38/Jun/FasL signaling and stabilization of p53, caus-
ing an increase in Bax and cytochrome c release, leading
to the activation of caspase-3 [15]. Delphinidin
3-sambubioside from Hibiscus induces apoptosis via
PARP inactivation; activation of caspase-3, -8, and -9;
and generation of reactive oxygen species in leukemia
[17]. Hibiscus anthocyanin-rich extract induces apoptosis

Selective Cytotoxicity and Apoptogenic Activity of Hibiscus Sabdariffa Aqueous Extract Against MCF-7 Human 397
Breast Cancer Cell Line
(a)
(b)
(c)
Figure 1. Selective cytotoxic activity of HSE against MCF-7 and HFFF cell lines. HFFF and MCF-7 cells were seeded, and
after 12 hours, increasing doses of HSE extract were added to the media. Cells were harvested after 24, 48, and 72 hours, and
cell numbers were analyzed using an MTT assay. No significant cytotoxic effect was observed in the MCF-7 cell line after 24
hours in all concentrations (1-a). There was significant reduction of cell numbers in the MCF-7 cell line following 48 and 72
hours in doses of 0.4 mg/ml and above (1-b, 1-c). At concentration of 0.5 mg/ml of the extract and following 72 hours incuba-
tion, cell survival was 45.51% (p < 0.05) (1-c).

Selective Cytotoxicity and Apoptogenic Activity of Hibiscus Sabdariffa Aqueous Extract Against MCF-7 Human
Breast Cancer Cell Line
398
HFFF MCF-7 Size Marker
Figure 2. Apoptogenic activity of HSE against MCF-7 and HFFF cell line. To check for DNA fragmentation as a sign of apop-
tosis, DNA was purified and analyzed on agarose gel. At the dosage of 0.5 mg/ml following 72 hours of incubation, clear for-
mation of a DNA ladder was observed in the MCF-7 cell line. No ladder formation was observed in HFFF cells.
in promyelocytic leukemia cells by increased phos-
phorylation of p38 and c-Jun, cytochrome c release, and expression of tBid, Fas, and FasL [18]. Induction of apoptosis by Hibiscus protocatechuic acid in human leu-

Selective Cytotoxicity and Apoptogenic Activity of Hibiscus Sabdariffa Aqueous Extract Against MCF-7 Human 399
Breast Cancer Cell Line
kemia cells via reduction of retinoblastoma phosphoryla-
tion and Bcl-2 expression is also reported [19].
Previous investigators have shown that aqueous ex-
tract of Hibiscus sabdariffa might contain anthocyanins,
polyphenols, and flavonoids [12]. Flavonoids [20],
polyphenols [21,22], and anthocyanins [23,24] are all
shown to have anticancer effects. Meanwhile, we have
not yet specifically looked into the active ingredient(s) of
the HSE that inhibits the growth of breast cancer.
Use of inappropriate concentrations of dietary phyto-
chemicals in mechanistic studies may generate artifactual
results, which can be misleading and physiologically
irrelevant [25]. We have observed cytotoxicity of the
crude extract at doses below that reported by other inves-
tigators in other cancer cell typ es [14], which implies that
breast cancer is a better model for in vivo studies of HSE
because of a more realistic predicted in vivo concentra-
tion.
Forty to seventy percent of breast tumors are estrogen
receptor positive, and considerable effort has been tar-
geted towards blocking estrogen receptors for treatment
and chemoprevention of breast cancer [26]. Phyto- oes-
trogens were originally proposed as cancer protective
agents following epidemiological observations revealing
a low breast cancer incidence in soy-consuming popula-
tions [27]. There are reports that HSE might have estro-
genic effects, although the exact estrogen-like ingredient
is not determined [12]. HSE contains at least two phy-
tosterols, β-sitosterol and ergosterol [12]. Ergosterol has
been previously shown to have apoptogenic effect
against MCF-7 cell lines [28]. Oral β-sitosterol markedly
reduces E2-induced MCF-7 tumors in mice [29] and
lowers serum estrogen levels, suggesting that consump-
tion of phytosterols from HSE may be beneficial for
women with breast cancer. We are interested in this as-
pect of HSE and plan to evaluate its effect on estrogen
receptors in breast carcinoma models.
While our results are promising regarding the antican-
cer effects of a relatively safe substance [12], use of
every crude herb is complicated by the lack of data re-
garding the consumption amount necessary to have the
desired effects in vivo.
5. Conflict of Interest
There is no conflict of interest with regard to this manu-
script.
6. Acknowledgements
The authors wish to express their gratitude to Tehran
University of Medical Sciences, which provided the
funding for this work (Grant Number: 86- 01-30-5355).
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